Antagonistic Effects of Oxidized Low Density Lipoprotein and α-Tocopherol on CD36 Scavenger Receptor Expression in Monocytes

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by α-tocopherol. We show here that α-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, α-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3′-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by α-tocopherol. However, α-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-γ (PPARγ; troglitazone), indicating that this pathway is susceptible to α-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and α-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARγ element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARγ and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARγ signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.

In many cell types, oxidized low density lipoproteins (oxLDL) 2 modulate cellular processes such as apoptosis, adhesion, migration, gene expression, and the induction of signal transduction cascades (1). Exposure of monocytes to oxLDL may alter gene expression and signaling, making them more susceptible to the following pro-atherogenic stimuli. The migration of monocytes into the intima and the conversion of monocytes/macrophages into foam cells represent initial steps in atherosclerosis. Current strategies to prevent atherosclerosis are aimed either at lowering the cholesterol load of lipoproteins or at reducing oxidative stress.
Vitamin E is a redox-active natural compound that can act, depending on the conditions, as a pro-or antioxidant on low density lipoproteins (LDL) in vitro and in vivo (2)(3)(4)(5). The major form of vitamin E in human plasma is ␣-tocopherol, and reduced plasma levels of ␣-tocopherol, such as in vitamin E-deficient mice, increase the incidence of atherosclerosis (6). Animal and cell culture studies strongly suggest that vitamin E can prevent atherosclerosis; however, the anti-atherogenic effects in clinical trials are still controversial (7)(8)(9)(10). ␣-Tocopherol in lipoproteins (mainly LDL) and also in the subendothelial space has been assumed to play a central role in reducing atherosclerosis by preventing lipid peroxidation and consequent lesion development. Nevertheless, since many compounds exist that can interfere with the oxidation of LDL without being equally effective, alternative modes of action have been proposed for atherosclerosis prevention, such as modulation of gene expression and cell signaling (reviewed in Refs. 7, 9, 11, and 12).
For vitamin E, non-antioxidant activities have been described, such as inhibition of vascular smooth muscle cell (VSMC) proliferation via inhibition of the protein kinase C (PKC) pathway; the modulation of phospholipase A 2 , cyclooxygenase-2, and 5-lipoxygenase and the release of interleukin-1␤; the reduction of cholesterol ester formation and uptake; the prevention of inflammation and monocyte/macrophage adhesion to the endothelium; the induction of connective tissue growth factor expression possibly involved in plaque stabilization (13); and the inhibition of scavenger receptor expression in smooth muscle cells and macrophages (14 -17).
The uptake of modified LDL leading to foam cell formation is mediated by scavenger receptors (class A; class B, type I; and CD36) (18). Expression of some scavenger receptors is increased at the atherosclerotic lesion (19), possibly as a result of a positive feedback loop mediated by oxLDL and its lipid content (19 -22). These receptors play also a major role in the uptake of vitamin E from high density lipoproteins in brain capillary endothelial cells and type II pneumocytes (23,24).
In addition to oxLDL, CD36 binds to a large variety of ligands: thrombospondin, collagen types I and IV, ␤-amyloid, fatty acids, anionic phospholipids, and high density lipoproteins (25). In various tissues, the uptake of long chain fatty acids is mediated by CD36/fatty acid translocase, and transgenic mice overexpressing CD36 have reduced blood lipids (26 -30). The central involvement of CD36 in atherosclerosis has been demonstrated by generating CD36 knockout mice, which show reduced uptake of modified LDL and reduced atherosclerosis (28). Similarly, human monocytes/macrophages from CD36-deficient patients show a low capacity to bind and internalize oxLDL (28,31); these mono-cytes show also decreased NF-B activation after oxLDL stimulation, leading to lower expression of inflammatory cytokines (32).
At the transcriptional level, CD36 expression is induced by oxLDL via the peroxisome proliferator-activated receptor-␥ (PPAR␥) and NF-E2related factor-2 (Nrf2) (21,(33)(34)(35). Activation of CD36 by interleukin-4, 15-deoxy-⌬ 12,14 -prostaglandin J 2 , and oxLDL in murine macrophages is dependent on PKC and PPAR␥ (36), whereas in THP-1 cells, induction by retinoic acid is independent of PPAR␥ and PKC (37). Cholesterol and cholesterol acetate increase CD36 expression possibly via activation of SREBP-1/2 and subsequent activation of PPAR␥ (38,39). Another protein kinase that was shown previously to be activated by oxLDL is protein kinase B (PKB). In VSMC, activation of PKB by oxLDL induces proliferation (40); and in mouse bone marrow-derived macrophages, it increases survival by preventing apoptosis (41). PKB has a wide range of cell targets, and its increased activity can be found during atherosclerosis and tumorigenesis (42). Activation of PKB involves a membrane translocation step, followed by phosphorylation of two key regulatory sites, Ser 473 and Thr 308 . After phosphorylation of PKB at Ser 473 by a yet unidentified kinase (PDK2), the enzyme becomes fully active (43,44).
We show here that vitamin E-deficient rats have increased expression of CD36 mRNA, indicating specific molecular mechanisms and signaling pathways modulated in vivo by ␣-tocopherol. In THP-1 monocytes, expression of the CD36 scavenger receptor was increased by oxLDL, and ␣-tocopherol treatment normalized it at both the protein and mRNA levels. PKC was not involved in the observed effects, but overexpression of PKB and PPAR␥ led to additive induction of CD36 promoter activity. Phosphorylation of PKB was increased by oxLDL, and ␣-tocopherol prevented it. Hence, our results suggest the existence of signaling pathways involving PPAR␥ and PKB that regulate CD36 expression in response to oxLDL and ␣-tocopherol in THP-1 monocytes.
CD36 Determination in Vitamin E-deficient Rats-Male Sprague-Dawley rats were housed five in a cage. The temperature was maintained between 21 and 22°C with a dark/light cycle of 12/12 h and with the lights turned on from 6.00 to 18.00 h. Three-month-old rats were randomly divided into two dietary groups: control rats fed ad libitum with a standard pellet diet (Harlan Italy) and rats fed a vitamin E-deficient diet. Fresh water was available ad libitum. During the experiment, the body weights of the vitamin E-deficient rats were unchanged; however, the rats had generally increased levels of urinary aldehydes (malondialdehydes, acetaldehydes, formaldehydes, propionaldehydes, and acetone) compared with control animals (data not shown). Tissue samples were taken in the morning under anesthesia with Nembutal (50 mg in 1 ml of saline/kg of body weight) following a 16-h (overnight) fasting period. The vitamin E levels (nmol/g of wet tissue) in the livers of the sacrificed rats were as follows: control group at the start of the experiment, 3 months old, 32.4 Ϯ 2.92; control group, 21-24 months old, 71.0 Ϯ 6.76; and vitamin E-deficient group, 21 months old, 1.6 Ϯ 0.25. The samples were stored in RNAlater reagent (Qiagen Inc.) at Ϫ80°C and later homogenized using a Polytron. Total tissue RNA was isolated using an RNA extraction kit (Qiagen Inc.). Determination of rat CD36 mRNA expression by reverse transcription (RT)-PCR was done as described and quantified after normalization to rat glyceraldehyde-3phosphate dehydrogenase mRNA expression (52).
Determination of CD36 mRNA by RT-PCR-Total RNA was isolated using the RNA extraction kit. Semiquantitative assays for CD36 mRNA expression were performed as described previously (15,53).
Determination of Total CD36 Expression by Western Blotting-Protein extraction and Western blotting were done according to standard methods with mouse anti-human CD36 monoclonal primary antibody (Ancell Corp.) and sheep anti-mouse IgG secondary antibody coupled to horseradish peroxidase (Amersham Biosciences) (55). Anti-␤-actin antibody (Sigma) was used as an internal control. Western blotting for PKB was done according to the protocol provided (Cell Signaling Technology). Proteins were visualized using an ECL detection kit (Amersham Biosciences) according to the manufacturer's recommendations. Chemiluminescence was monitored by exposure to Hyperfilm ECL film (Amersham Biosciences), and the signals were analyzed using a Lumi-Imager (Roche Applied Science).
Labeling and Uptake of oxLDL-Labeling of oxLDL with 3,3Ј-dioctadecyloxacarbocyanine perchlorate (DiO; Molecular Probes) and blocking of oxLDL-DiO uptake with anti-CD36 antibody were done as described previously (15). The uptake and binding of oxLDL-DiO were studied by FACS. The cells were pretreated for 16 h with 20 g/ml oxLDL, 50 mol/liter ␣-tocopherol, or 0.1% ethanol (solvent control) and then incubated with oxLDL-DiO (5 g/ml of medium). For the uptake experiments, incubation was carried out at 37°C for 6 h; and for the binding experiment, incubation was performed at 4°C for 30 min. Thereafter, the cells were washed three times with phosphate-buffered saline and 3% bovine serum albumin and once with phosphate-buffered saline and fixed with 4% paraformaldehyde in phosphate-buffered saline. FACS was performed with a FACScan, and data were analyzed using CellQuest software.
Plasmids, Transfections, and Reporter Assays-The firefly luciferase reporter plasmids used were pCD36 (15), pDR1 (56), and pNF-B (Clontech), and the Renilla internal control plasmid used was pRL-TK (Promega Corp.). The PKB expression vectors pPKBwt, pPKB(R25C), and pPKB(K179M) were kindly provided by Dr. J. Downward (Imperial Cancer Research Fund, London, UK) and correspond to pGFP-Akt, pGFP-Akt(R25C), and pGFP-Akt(K179M), respectively (57). The PPAR␥ expression vector used contains the PPAR␥ cDNA under the control of the cytomegalovirus promoter (56). THP-1 monocytes were transfected for 3 h with the indicated reporter and expression plasmids using Transfectin (Bio-Rad) and then treated with 0.1% ethanol (solvent control) or 50 mol/liter ␣-tocopherol for an additional 21 h. HEK293 cells were transfected for 3 h using Superfect (Qiagen Inc.). The medium was then changed, and the cells were treated with 0.1% ethanol (solvent control) or 50 mol/liter ␣-tocopherol and incubated for an additional 21 h. Extracts were prepared, and promoter activities were measured using the Dual-Luciferase assay kit (Promega Corp.) with a TD20/20 luminometer (Turner Designs). The promoter-firefly luciferase activities were normalized to the thymidine kinase promoter-Renilla luciferase activities, and the activities of the control transfections were set to 100%.
Apoptosis Assay-THP-1 cells were incubated with increasing concentrations of oxLDL for 24 and 48 h and then stained with Hoechst 33342 (5 g/ml) for 1 h at 37°C (58). A total of 200 cells for each treatment were counted, and the percentage of apoptotic cells with condensed nuclei was quantified.
Statistical Analysis-Values are expressed as the mean Ϯ S.D. as indicated in the figure legends. For FACS results, the median fluorescence intensity was determined, and the mean Ϯ S.D. was calculated as described in the figure legends. Student's t test was used to analyze the significant differences between two conditions. A p value Ͻ0.05 was taken as significant and is indicated by asterisks in the figures.

Vitamin E Deficiency in Rats Leads to CD36 Overexpression-Our
previous in vitro results suggested that, in VSMC and HL-60 cells, the expression level of CD36 is inhibited by ␣-tocopherol, leading to reduced uptake of oxLDL (15). To assess whether a similar regulation takes place also in vivo, rats were deprived of ␣-tocopherol intake for 21 months, and the expression level of CD36 mRNA was measured by RT-PCR. ␣-Tocopherol deficiency led to a significantly higher level of liver CD36 expression (238 Ϯ 41% (n ϭ 4); p Ͻ 0.028) compared with rats kept on a normal diet (set to 100 Ϯ 38% (n ϭ 4)). The increased level of urinary aldehydes observed in vitamin E-deficient animals (data not shown) suggests increased oxidative stress; thus, ␣-tocopherol could inhibit CD36 expression by reducing oxidative stress or, alternatively, by interfering with signal transduction and gene expression modulated by oxidized molecules.
␣-Tocopherol Inhibits CD36 Surface Overexpression Induced by oxLDL in THP-1 Monocytes-The in vivo results described above could be explained by increased generation of oxLDL in vitamin E-deficient animals, an event that possibly could be prevented by supplementation with ␣-tocopherol. On the other hand, it has been shown that ␣-tocopherol can reduce CD36 expression in VSMC and monocytes/ macrophages by interfering with signal transduction and CD36 gene expression (15,17).
To delineate these two pathways affected by ␣-tocopherol, CD36 expression was induced by oxLDL in THP-1 monocytes, and we checked whether ␣-tocopherol could interfere with this pro-atherogenic stimulus. By using oxLDL in this model system, a reduction of CD36 overexpression by ␣-tocopherol would be the consequence of inhibition of oxLDL-induced CD36 transcription rather than the result of inhibition of LDL oxidation.
Treatment with ␣-tocopherol in the absence of oxLDL stimulation did not reduce CD36 expression in THP-1 monocytes as described previously (53) and was therefore specifically antagonizing only upon oxLDL stimulation. Neither LDL nor aggregated LDL increased CD36 expression in this cell line (data not shown).
Apoptosis Induction Only at High oxLDL Concentrations-Treatment with oxLDL is also known to induce apoptosis, and it seemed possible that the observed increase in CD36 expression was a consequence of cell death and cellular toxicity. However, when THP-1 cells were incubated for 24 and 48 h with increasing concentrations of oxLDL (10 -80 g/ml), significant apoptosis as measured by assessing the number of condensed nuclei was observed only with the highest concentration (80 g/ml) and after 48 h (Fig. 2). Taken together, these results suggest that oxLDL induce CD36 expression by activating a signaling cascade and that ␣-tocopherol can interfere with this event.
␣-Tocopherol Inhibits CD36 Overexpression at the Protein and mRNA Levels in THP-1 Monocytes Stimulated by oxLDL-The results obtained by FACS could be explained by increased gene and protein expression of CD36. Western blotting with anti-CD36 monoclonal antibody showed that CD36 protein is expressed in THP-1 monocytes mainly as a 74-kDa protein as described previously (59). Treatment of THP-1 monocytes with oxLDL (20 g/ml) stimulated total CD36 protein expression, and co-treatment with ␣-tocopherol (50 mol/liter) prevented CD36 overexpression completely (reduction of 71 Ϯ 38%) in Western blot analyses (Fig. 3).
To assess whether modulation of CD36 protein expression by oxLDL and ␣-tocopherol in THP-1 monocytes is the result of changes in gene expression, THP-1 monocytes were incubated with oxLDL (20 g/ml) in the presence of ␣-tocopherol (50 mol/liter) or ethanol (0.1%; solvent control), and CD36 mRNA expression was analyzed by RT-PCR. Treatment with oxLDL led to increased expression of CD36 mRNA, whereas co-treatment with ␣-tocopherol normalized CD36 mRNA levels (reduction of 39 Ϯ 28%) (Fig. 4). Thus, in THP-1 monocytes, oxLDL stimulates CD36 gene and protein expression, effects that are prevented by ␣-tocopherol.
Uptake and Binding of oxLDL-DiO Are Inhibited by ␣-Tocopherol in THP-1 Monocytes Stimulated by oxLDL-Expression of the CD36 scavenger receptor is involved in the uptake of oxLDL, allowing the accu- mulation of lipids and cholesterol that ultimately lead to foam cell formation. Consequently, inhibition of CD36 expression should reduce oxLDL uptake. To check this hypothesis, THP-1 monocytes were incubated with ␣-tocopherol (50 mol/liter) or ethanol (0.1%; solvent control), and the uptake or binding of fluorescently labeled oxLDL-DiO was analyzed by FACS. In unstimulated THP-1 monocytes, treatment with ␣-tocopherol resulted in a small and non-significant decrease in oxLDL-DiO binding and uptake (Fig. 5A). In oxLDL-stimulated cells, ␣-tocopherol treatment led to stronger and statistically significant inhibition of oxLDL binding and uptake (Fig. 5B).
To check whether CD36 is the major scavenger receptor responsible for the uptake of oxLDL, THP-1 monocytes were preincubated for 30 min with anti-CD36 monoclonal antibody before addition of oxLDL-DiO for 6 h. The uptake of oxLDL-DiO was analyzed by FACS. As described previously for other cell types (15,49,60,61), anti-CD36 antibody interfered with the uptake of oxLDL-DiO, whereas an isotypematched control antibody (mouse anti-IgM antibody) did not (data not shown), suggesting that CD36 is responsible for oxLDL uptake in THP-1 monocytes.
Modulation of CD36 Expression in THP-1 Monocytes by oxLDL and ␣-Tocopherol Does Not Involve PKC-In VSMC, ␣-tocopherol inhibits PKC by activation of protein phosphatase 2A, leading to inhibition of proliferation (62). Because oxLDL are known to activate PKC in THP-1 macrophages (63), it was possible that the above-described inhibitory effect on CD36 expression mediated by ␣-tocopherol was the result of PKC inhibition.
The involvement of PKC in CD36 modulation was investigated by treating THP-1 monocytes with an activator of PKC (PMA) and a specific PKC inhibitor (PKC412). CD36 expression was analyzed by FACS.
When THP-1 monocytes were differentiated to macrophages by treatment with PMA (5 nmol/liter) for 24 h, CD36 was induced at the mRNA level as analyzed by RT-PCR and at the protein level as analyzed

. Expression of CD36 mRNA induced by oxLDL is inhibited by ␣-tocopherol.
A, after treatment of THP-1 monocytes as indicated, total RNA was isolated, and CD36 mRNA expression was quantified by RT-PCR. B, the graph shows the CD36 signal normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each bar represents the mean Ϯ S.D. of two independent experiments. *, p Ͻ 0.05 versus the control set to 100%. E, ethanol (solvent control); T, ␣-tocopherol.
Modulation of CD36 Expression in THP-1 Monocytes by oxLDL and ␣-Tocopherol Involves PPAR␥-The CD36 scavenger receptor has been described to be strongly activated by PPAR␥, a nuclear receptor responsive to lipid peroxidation products present in oxLDL. In PMA-stimulated THP-1 cells, CD36 is inhibited completely by GW9662, an irreversible PPAR␥ antagonist (37); and troglitazone, a specific PPAR␥ agonist, induces CD36 expression in mouse peritoneal macrophages (64).
To determine whether the CD36 promoter is activated at the transcriptional level, THP-1 monocytes were transfected with a CD36 promoter-luciferase reporter vector containing 380 bp of the human CD36 promoter (pCD36) (15) and treated with oxLDL (20 g/ml) or troglitazone (50 mol/liter). Furthermore, to assess whether the observed modulation by oxLDL or troglitazone occurred via PPAR␥ activation, a reporter plasmid containing a PPAR␥-responsive element that controls a thymidine kinase basic promoter (pDR1) was transfected into THP-1 cells and treated as described above. Activation of the pCD36 and pDR1 constructs with similar potency by oxLDL and troglitazone suggests that these compounds stimulate the CD36 promoter via activation of PPAR␥ (Fig. 8, A and B). In line with the above-described results obtained by FACS (Fig. 1), Western blotting (Fig. 3), and RT-PCR (Fig.  4), co-treatment with ␣-tocopherol (50 mol/liter) partially reduced CD36 overexpression induced by oxLDL as well as by troglitazone (Fig. 8C).
Altogether, these results led to the conclusion that oxLDL and ␣-tocopherol act antagonistically on the PPAR␥ signaling pathway in THP-1 monocytes, without direct involvement of PKC. This is in line with previous experiments in which PPAR␥ activity was up-regulated by oxLDL in a PKC-independent manner (65).
oxLDL-induced PKB Phosphorylation Is Prevented by ␣-Tocopherol-Another protein kinase that was shown previously to be activated by oxLDL is PKB. Interestingly, in human HMC-1 mastocytoma cells, PKB phosphorylation is inhibited by tocopherols (66), suggesting that the observed effects of oxLDL and ␣-tocopherol on CD36 expression could be the result of modulation of PKB activity.
PKB Activates CD36 Expression and Stimulates PPAR␥ and NF-B Activities-It has furthermore been shown that activation of the PKB pathway by platelet-derived growth factor leads to induction of PPAR␥ gene expression in VSMC (67,68), suggesting that activation of PKB by oxLDL may be involved in activation of PPAR␥/CD36 expression. In line with this, oxLDL-induced expression of scavenger receptors (class A, type I; class B, type I; and CD36) is prevented by treatment with specific phosphatidylinositol 3-kinase inhibitors that reduce PKB activation (41,69).
The ability of PKB to activate CD36 via the PPAR␥ element in its promoter was investigated by cotransfection of the PKB expression vectors with a luciferase reporter plasmid carrying a binding site for this FIGURE 6. PKC is not involved in CD36 modulation by oxLDL and ␣-tocopherol. PKC was stimulated by PMA for 24 h, which led to adhesion of THP-1 monocytes and differentiation. A, CD36 mRNA expression was induced to 205 Ϯ 65% (n ϭ 3) as analyzed by RT-PCR. *, p Ͻ 0.048 versus the control set to 100%. B, CD36 protein expression was induced to 527 Ϯ 17% (n ϭ 4) as analyzed by FACS. *, p Ͻ 0.005 versus the control set to 100%. In both cases, ␣-tocopherol co-treatment did not reduce CD36 expression. C, THP-1 monocytes were stimulated with oxLDL (20 g/ml) in the presence or absence of ␣-tocopherol for 24 h. Additional treatment with PKC412 (1 mol/liter) did not lead to further inhibition of CD36 expression (n ϭ 3). E, ethanol (solvent control); T, ␣-tocopherol; FITC, fluorescein isothiocyanate.
transcription factor (pDR1). In addition to this, as control, the effect of PKB expression was checked using a reporter vector carrying a site for NF-B (pNF-B), known to be activated by PKB. Overexpression of wild-type PKB, but not mutant inactive PKB, activated pDR1 and pNF-B, suggesting that both PPAR␥ and NF-B elements are up-regulated by PKB in THP-1 cells (Fig. 10A).
PKB Acts on PPAR␥ to Induce CD36 Expression-Because cotransfection experiments with multiple vectors were difficult to perform in THP-1 cells, the effects of PKB and PPAR␥ expression were further assessed in HEK293 cells, which can be transfected with higher efficiency. Similar to THP-1 cells, wild-type PKB, but not mutant PKB, induced CD36 expression (pCD36) in parallel with activation of the PPAR␥ (pDR1) and NF-B (pNF-B) elements in HEK293 cells (Fig. 10B).
To determine whether PKB can activate PPAR␥, pPKBwt or mutant pPKB was cotransfected with a PPAR␥ expression vector (pPPAR␥) into HEK293 cells, and pCD36, pDR1, and pNF-B activities were measured. Overexpression of PPAR␥ induced CD36 expression and stimulated the PPAR␥ element, but not the NF-B element, which was activated only by wild-type PKB (Fig. 10C). Interestingly, the effect of combined PKB and PPAR␥ expression on CD36 expression as well as on the PPAR␥ element was additive (Fig. 10C). These results suggest that CD36 expression is activated by PKB via the previously described PPAR␥ element in its promoter sequence (33,55).

DISCUSSION
In this study, we investigated whether ␣-tocopherol (the most abundant form of vitamin E in human plasma) acts at the earliest events of the cascade of atherosclerosis progression: oxLDL binding and uptake by monocytes. We have shown that ␣-tocopherol prevented oxLDLinduced CD36 overexpression and reduced the binding and uptake of oxLDL. Activation by oxLDL was always required for the inhibitory action of ␣-tocopherol. Thus, oxLDL may stimulate THP-1 monocytes by binding to membrane receptors such as CD36, which may trigger alterations in cell signaling.
PKC is known to be activated by oxLDL and inhibited by ␣-tocopherol, but our results with PMA and PKC412 do not support the involve-   . oxLDL-stimulated PKB phosphorylation at Ser 473 is inhibited by ␣-tocopherol. THP-1 monocytes were treated with oxLDL (20 g/ml) and either ethanol (0.1%; solvent control) or ␣-tocopherol (50 mol/liter) for 24 h. Upper panel, Western blot of total protein isolated from THP-1 monocytes. The blot was probed with anti-phospho-Ser 473 PKB antibody and subsequently with anti-PKB antibody. Anti-␤-actin antibody was used as a control. Lower panel, graphical display of the relative PKB phosphorylation of the Western blots. Treatment with oxLDL induced phosphorylation of PKB at Ser 473 to 135 Ϯ 15% (n ϭ 3; *, p Ͻ 0.001 versus the control set to 100%), and ␣-tocopherol reduced it to 72 Ϯ 12% (n ϭ 3; **, p Ͻ 0.0003 versus oxLDL-treated). E, ethanol (solvent control); T, ␣-tocopherol. ment of PKC in the inhibition of CD36 expression by ␣-tocopherol. Several transcription factors have been described that are modulated by ␣-tocopherol by direct binding (e.g. pregnane X receptor (PXR) and possibly other nuclear receptors) (70) or by changing their activity (such as AP-1 or NF-B) (reviewed in Ref. 12). We found that, similar to oxLDL, troglitazone activates PPAR␥ (71,72) with consequent increased CD36 expression that can be partially normalized by ␣-tocopherol. Thus, the reduction of CD36 expression by ␣-tocopherol occurs via the PPAR␥ signaling pathway and is not mediated by the PKC pathway.
␣-Tocopherol may modulate PPAR␥ activity by affecting its redox state (73), its phosphorylation by mitogen-activate protein kinase (74), the rate of its synthesis (75), or proteolytic degradation (76). Moreover, ␣-tocopherol could also interfere with the action of lipid peroxidation products that were found to be increased in vivo in vitamin E-deficient rats and that may, after being internalized with oxLDL, increase CD36 expression via PPAR␥ activation. However, a recent study indicates that, although aldehydes can induce CD36 expression in THP-1 monocytes, ␣-tocopherol only partially reduces it, suggesting additional signaling pathways (77).
Our results show that oxLDL stimulates PKB phosphorylation, an event that can be inhibited by ␣-tocopherol. Because PKB stimulates PPAR␥ activity with consequent CD36 promoter activation, it is likely that ␣-tocopherol affects oxLDL-stimulated CD36 expression via inhibition of PKB phosphorylation. In hepatocytes, increased PKB activity has been shown to activate SREBP-1 (78), which activates PPAR␥ in adipocytes by the production of endogenous ligands (79), and it remains to be shown whether a similar activation cascade is functional in THP-1 monocytes.
The tocopherols were recently described to interfere with PKB phosphorylation at Ser 473 , leading to reduced proliferation of HMC-1 mast cells (66). In other cell lines such as breast cancer cells, PKB phosphorylation is inhibited by tocotrienols after stimulation by epidermal growth factor (80) and also by the two tocopherol derivatives ␣-tocopheryl succinate and ␣-tocopheryloxybutyric acid (81). Further studies have shown that ␥-tocotrienol induces a large decrease in the relative intracellular levels of the phosphorylated forms of PDK1, PKB, and glycogen synthase kinase-3 (82,83).
The tocopherols and tocotrienols may inhibit PKB phosphorylation at Ser 473 either directly or by acting on enzymes upstream of PKB, such as receptor tyrosine kinases (Tyk2) (17), phosphatidylinositol 3-kinase, and a kinase phosphorylating PKB (PDK1/2) (Fig. 11). Alternatively, ␣-tocopherol may stimulate a phosphatase dephosphorylating phospho-PKB such as protein phosphatase 2A or a lipid phosphatase (pTEN) that hydrolyzes the products of phosphatidylinositol 3-kinase (84,85). In addition, the tocopherol-associated proteins, which modulate phosphatidylinositol 3-kinase and PKB in vitro and in vivo, may also play a role in the observed effects of ␣-tocopherol on CD36 expression in THP-1 monocytes (86,87).
Several studies indicate that tyrosine phosphorylation is modulated by the tocopherols. In oxLDL-stimulated MRC5 fibroblasts, tyrosine phosphorylation of JAK2, STAT1 (signal transducer and activator of transcription), and STAT3 is reduced by ␣-tocopherol (88). In VSMC, angiotensin II-induced tyrosine phosphorylation of two major proteins (p120 and p70) and ERK (extracellular signal-regulated kinase) activation are markedly reduced by ␣-tocopherol, whereas ERK activation by epidermal growth factor is unaffected (89). Tyrosine phosphorylation is also decreased by ␣-tocopheryl succinate in human neutrophils via activation of a tyrosine phosphatase (90). Because class I and II phosphatidylinositol 3-kinases are regulated by tyrosine phosphorylation, it can be speculated that inhibition of tyrosine kinase activity by tocopherols may ultimately lead to reduced PKB membrane translocation and phosphorylation (91).
When extrapolated to other cell types, our results suggest that activation of PKB leads to increased CD36 expression, ultimately increasing the uptake of fatty acids. Activation of PKB also occurs in response to insulin, which stimulates glucose uptake by increasing glucose transporters (GLUT-4 and GLUT-1) and by stimulating glucokinase expression (92,93). Energy from glucose is used for the biosynthesis of fatty acids and cholesterol. SREBP-1 and PPAR␥ are the major transcription factors regulating these processes, and both are activated by PKB (39,79,94,95). Thus, the finding that PKB increases CD36 expression suggests that increased fatty acid synthesis by activating SREBP-1 is assisted by increased fatty acid uptake by activating PPAR␥ and subsequent activation of CD36 expression (96).
In the presence of insulin (e.g. postprandial), increased CD36 expression may be involved in removal of fatty acids from plasma and also in regulation of insulin secretion by fatty acids in pancreatic ␤-cells (97). However, in the absence of insulin (e.g. diabetes) or during impaired insulin signaling (e.g. insulin resistance), a lower activation of CD36 expression may lead to insufficient plasma lipid removal with consequent hyperlipidemia (29). In this situation in which cells are exposed to increased lipid concentrations, oxLDL and possibly glucose-oxidized LDL may further accelerate lipid uptake via CD36 overexpression consequent to activation of the PKB/PPAR␥ pathway (98).
In summary, our findings show that ␣-tocopherol reduces the cellular effects of oxLDL by interfering with CD36 gene and protein expression, and our data suggest that PKB and PPAR␥ are involved in this process. Thus, ␣-tocopherol may have a beneficial role not only in tissue macrophages, but already at earlier times such as during plasma and tissue monocyte activation. Recently, it was shown that one of the first steps in atherogenesis, the adhesion of THP-1 monocytes, is inhibited by ␣/␥-tocopherol and ␣/␥-tocotrienol in tumor necrosis factor-␣-activated human umbilical vein endothelial cells (99). It remains to be shown whether ␣-tocopherol influences further early atherosclerotic events induced by oxLDL in monocytes, such as rolling on the endothelium, migration into the intima, and subsequent differentiation to macrophages (100).